Proteomic analysis of macrophage responses to distinct collagen type I morphologies
Cellular and molecular mechanisms (English)Pulmonary fibrosis is the end-stage of multiple lung diseases, characterized by scarring of the lungs and loss of function as a result. Macrophages are known to be important players in tissue homeostasis, but their ability to respond to fibrosis-related morphological and mechanical changes of the extracellular matrix (ECM) is relatively unexplored. Our previous work indicated that murine alveolar-like macrophages are sensitive to the morphology of collagen type I in vitro. Higher expression of Ym1, a murine marker of pro-healing M2 macrophages, was found when macrophages were cultured on fibrous collagen. Globular collagen led to higher expression of CD206 (the mannose receptor), a marker known to be upregulated on alveolar macrophages in idiopathic pulmonary fibrosis. Moreover, macrophages exhibited distinct differences in shape in response to fibrous and globular collagen, with a more amoeboid appearance on fibrous collagen and a more mesenchymal appearance with many filopodia on globular collagen. In parallel to these alterations in macrophage shape, transmigration was higher when macrophages were cultured on fibrous collagen compared to the uncoated condition. Currently, we are applying differential proteomics to further investigate the molecular mechanisms behind these changes in marker expression, shape and behavior of macrophages. To generate a macrophage-specific protein/peptide library, we separated the tryptic peptides of macrophage protein lysates and analyzed the fractions by LC-MS/MS using a high-resolution orbitrap mass spectrometer. The generated library consisted of 7.031 proteins with at least one unique peptide for each protein and 33.291 unique peptides. Our macrophage library now covers 41% of the theoretical mouse proteome and we expect to expand this in future experiments. Next, the protein lysates of macrophages cultured on uncoated, fibrous collagen-coated or globular collagen-coated substrates were trypsin-digested to peptides and analyzed by LC-MS. After data processing using the macrophage-specific protein library, 2.117 unique proteins were reproducibly quantified in the three different conditions. Currently, we are analyzing our data to identify condition-specific protein profiles and to unravel which pathways are involved in the observed macrophage responses to different collagen morphologies. This study emphasizes the complexity of interactions between macrophages and their surroundings, and stresses the need to investigate the role of macrophage-matrix interactions in pulmonary fibrosis.